The goal of this project is to determine the key factors that regulate NO synthesis by nitric oxide synthase (NOS). Three major isoforms of NOS has been discovered: eNOS from endothelia, nNOS from neurons and iNOS from macrophages. NOS is a dimeric enzyme that is comprised of two domains, a heme-containing oxygenase domain, where arginine is oxidized to citrulline and NO, and a reductase domain, which transfers electrons from NADPH to the heme active center. Each expressed oxygenase domain, which consists of a heme group, a tetrahydrobiopterin cofactor (H4B) and the substrate binding site, will be used as models for the full length enzymes in this project. In the current grant period, we have discovered that binding of H4B to NO-bound protein significantly distorts the heme macrocycle thereby altering the heme redox potential, and that NO auto-inhibits the enzyme and disrupts the dimeric interactions. In addition, we found that the H-bond between a tryptophan and the proximal cysteine that coordinates to the heme iron is very important in modulating the electronic properties of the iron-cysteine bond, which is critical for NO synthesis. We also characterized the first oxygen intermediate during NOS catalysis with resonance Raman spectroscopy. To further elucidate the mechanistic details of NOS function, four (4) series of experiments are proposed. First, the iron-sulfur stretching mode of the heme proximal bond will be systematically studied to evaluate its impact on the catalytic activity. Second, a novel microfluidic mixer coupled with a rapid freeze quenching device that we have developed in our lab along with several mechanistic strategies will be employed to trap and identify the various oxygen intermediates in the catalytic cycle. Third, three (3) state-of-the-art EPR techniques will be used to characterize the H4B radicals formed during the reaction. Finally, the role of heme distortion on the catalytic properties of the enzyme and the NO-induced disruption of the dimeric interactions will be further studied to construct an integrated picture of the NO-mediated regulatory mechanisms. In this work, all three (3) NOS isoforms will be studied, and isoform specific properties will be systematically evaluated. The information generated from this project will serve as a foundation to improve our understanding of various disease states linked to NOS and will be very valuable in facilitating the rational design of isoform specific inhibitors.